The voltage dependence of the TMEM16B/anoctamin2 calcium-activated chloride channel is modified by mutations in the first putative intracellular loop.
The voltage dependence of the TMEM16B/anoctamin2 calcium-activated chloride channel is modified by mutations in the first putative intracellular loop.
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DOI：
10.1085/jgp.201110764
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发表时间：
2012-04
期刊：
影响因子：
--
通讯作者：
Menini A
中科院分区：
文献类型：
--
作者：
Cenedese V;Betto G;Celsi F;Cherian OL;Pifferi S;Menini A
钙激活的氯离子通道(CaCC)参与多种生理过程。最近，TMEM16A/anocTamin1和TMEM16B/anocTamin2被证明具有钙通道的功能，但关于这些通道的结构-功能关系的信息很少。TMEM16B表达于嗅觉感觉神经元的纤毛、犁鼻感觉神经元的微绒毛和视网膜光感受器的突触终末。在这里，我们首次对TMEM16B进行了定点突变研究，以了解电压和钙依赖的分子机制。我们在第一个可能的细胞内环中突变了氨基酸，并使用全细胞电压钳技术测量了在不同细胞内钙浓度存在下，野生型和突变型TMEM16B通道在HEK 293T细胞中表达的性质。我们将E367突变为谷氨酰胺或缺失5个连续的谷氨酸386EEE390和399EYE401。眼睛的缺失并没有显著改变通道激活的明显的钙依赖和电压依赖。E367Q和5个谷氨酸的缺失对钙离子的表观亲和力影响不大，但改变了电压依赖性，使电导-电压关系向更正的电压方向移动。这些发现表明，细胞内第一环中的谷氨酸E367和386EEE390在TMEM16B的电压依赖中起重要作用，从而为该通道的结构和功能提供了初步的研究。
Ca2+-activated Cl− channels (CaCCs) are involved in several physiological processes. Recently, TMEM16A/anoctamin1 and TMEM16B/anoctamin2 have been shown to function as CaCCs, but very little information is available on the structure–function relations of these channels. TMEM16B is expressed in the cilia of olfactory sensory neurons, in microvilli of vomeronasal sensory neurons, and in the synaptic terminals of retinal photoreceptors. Here, we have performed the first site-directed mutagenesis study on TMEM16B to understand the molecular mechanisms of voltage and Ca2+ dependence. We have mutated amino acids in the first putative intracellular loop and measured the properties of the wild-type and mutant TMEM16B channels expressed in HEK 293T cells using the whole cell voltage-clamp technique in the presence of various intracellular Ca2+ concentrations. We mutated E367 into glutamine or deleted the five consecutive glutamates 386EEEEE390 and 399EYE401. The EYE deletion did not significantly modify the apparent Ca2+ dependence nor the voltage dependence of channel activation. E367Q and deletion of the five glutamates did not greatly affect the apparent Ca2+ affinity but modified the voltage dependence, shifting the conductance–voltage relations toward more positive voltages. These findings indicate that glutamates E367 and 386EEEEE390 in the first intracellular putative loop play an important role in the voltage dependence of TMEM16B, thus providing an initial structure–function study for this channel.
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DOI：
10.1590/s0100-879x2009005000028
发表时间：
2009-11-01
影响因子：
2.3
作者：
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通讯作者：
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Galietta, Luis J. V.
影响因子：
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影响因子：
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